Ion channels mediate the flow of ions across cellular membranes during electrical excitation. Many neurotransmitters and hormones regulate ion channel activity through second messenger-activated phosphorylation. Although several second messenger-dependent protein kinases are known to directly phosphorylate ion channels, little is known about the protein phosphatases that terminate the influence of phosphorylation. The general goal of this proposal is to define the role of phosphatases in controlling the state of ion channel phosphorylation, and to determine whether phosphatase action on ion channels is also regulated by neurotransmitters or hormones. This proposal focuses on identifying the phosphatases and regulatory pathways controlling phosphorylation of voltage-sensitive sodium channels in rat brain. These channels are phosphorylated by cAMP-dependent protein kinase in situ. Cyclic AMP-dependent phosphorylation may modify the electrophysiological properties of neuronal sodium channels. Because its structure is well characterized and physiologically relevant phosphorylation sites have been identified, the voltage-sensitive sodium channel is an excellent candidate for studying dephosphorylation. Biochemical studies will be performed to identify the brain phosphatase(s) that dephosphorylates cAMP-dependent phosphorylation sites in the sodium channel. Slices from discrete regions of adult rat brain will be used to examine how this phosphatase is regulated in intact neurons and whether specific neurotransmitter pathways in the brain control cAMP-dependent phosphorylation or dephosphorylation of sodium channels. Finally, the regional and subcellular localization of the sodium channel phosphatase(s) will be examined using both biochemical and immunocytochemical methods, and the distribution of the enzyme(s) compared with that of sodium channels. By using this strategy, a detailed picture will be developed of how sodium channel phosphorylation is controlled in brain. This may yield new insight and biochemical tools with which to examine the functional consequences of sodium channel phosphorylation. In addition, this study will address the general question of whether phosphatases are regulated by neurotransmitters or hormones and how phosphatases participate in controlling the state of phosphorylation of ion channels and other neuronal phosphoproteins. Because reversible phosphorylation is such an important means of modulating ion channel function and consequently cellular excitation, it is important to learn how this complex regulatory system operates if we are to understand the role of neuronal excitability in health and disease.